6. Project Summary/Abstract: Limb trauma can lead to the development of a complex regional pain syndrome (CRPS). At onset this syndrome presents a baffling array vascular, bone, and nociceptive changes in the injured limb that can evolve into chronic edema, pain, weakness, contractures, and osteoporosis. Currently there is no consensus on either the pathophysiology or treatment for CRPS and there is a need for translational studies to identify therapeutic targets and novel pharmacologic approaches. The most common etiology for CRPS is distal limb fracture and we have developed a rat fracture model resembling CRPS. After distal tibia fracture and cast immobilization for 4 weeks the rats develop chronic unilateral hindlimb warmth, edema, increased spontaneous protein extravasation, periarticular osteoporosis, hindlimb unweighting, and allodynia, changes paralleling those observed in CRPS patients. The vascular and nociceptive changes characteristic of the early stages of CRPS suggest an inflammatory process, yet there is little evidence for an immunocyte response. On the other hand, there is compelling evidence that neurogenic inflammatory responses mediated by substance P (SP) signaling are exaggerated in the CRPS limb and in the rat fracture model. Substance P signaling stimulates keratinocyte proliferation, activation, and cytokine and growth factor production in vitro, including tumor necrosis factor alpha (TNF), interleukin-1 beta (IL-1), interleukin-6 (IL-6), and nerve growth factor (NGF). TNF and IL-6 are up-regulated in experimental skin blisters in CRPS limbs and we have observed that all these inflammatory cytokines and NGF are up-regulated in the hindpaw skin of the fracture rats. When we administered an SP receptor antagonist to the fracture rats the increase in cutaneous cytokines and NGF was prevented. Fracture rats treated with cytokine inhibitors or anti-NGF failed to develop allodynia and had attenuated hindlimb unweighting, indicating an important role for cytokine and growth factor signaling in the development of trauma induced pain. Based on these data we propose that fracture and immobilization enhance SP endothelial signaling in the injured limb, resulting in vascular inflammation with increased extravasation, edema and warmth. We also postulate that facilitated SP signaling activates keratinocyte expression of pronociceptive cytokines and NGF, resulting in post-traumatic pain symptoms. The primary objective of this proposal is to identify target-specific treatments for CRPS using approved or soon to be approved medications. This work is directed at understanding the peripheral sources of mediators supporting post-traumatic inflammation and pain sensitization and determining whether these mediators are associated with risk factors for the development of post-traumatic CRPS. We will also determine whether peripheral inflammatory mediators contribute to the transition of post-traumatic pain into a chronic pain state. The specific aims are; 1) to use immunohistochemistry methods to map post-traumatic changes in SP, inflammatory cytokine, and NGF signaling in keratinocytes, endothelial cells, and mast cells in the skin of CRPS patients and in a rat fracture model of CRPS, 2) identify risk factors for development of CRPS-like sequelae in the rat fracture model, including age, sex, ACE inhibitor usage, and immobility, and then use PCR, EIA, western blotting and extravasation assays to determine whether facilitated SP signaling and increased inflammatory cytokine and NGF expression are associated with an increased risk of developing post-traumatic CRPS in these conditions, and 3) establish the efficacy of the global cytokine inhibitor pentoxifylline, the SP receptor antagonist LY303870, and the NGF antibody tanezumab in preventing or attenuating the transition from early post-traumatic pain to chronic pain, and to evaluate their efficacy for treating chronic pain (16 weeks post-fracture) in the CRPS fracture model. These studies are expected to generate translational data supporting future clinical CRPS trials, with the potential for improving the efficacy and safety of the clinical management of post-traumatic pain syndromes. PUBLIC HEALTH RELEVANCE: 7. Project Narrative: The proposed studies in this application would provide a sound foundation for future clinical investigations establishing the chronic inflammatory basis of post-traumatic complex regional pain syndrome (CRPS) and the therapeutic efficacy of SP receptor antagonists, cytokine inhibitors and anti-NGF treatment. The treatments being investigated in these experiments are all FDA approved or are currently being developed in clinical trials, therefore, positive results in the CRPS fracture model could be readily translated into clinical CRPS trials. A new generation of war wounded veterans are now being seen in the VA Health Care System with blast injury induced traumatic injuries to the limbs and head. This proposal has the potential to identify novel pharmacologic treatments that could reduce pain and enhance the quality of life in war-wounded veterans.